Transcription of the S.cerevisiae HO gene occurs immediately after and is dependent upon the Start of the cell cycle. The promoter element CACGAAAA (or SCB) and two trans-acting regulators (SWI4 and SWI6) that are required for the cell cycle regulation of HO transcription have been identified (Breeden and Nasmyth, 1987a). These SCB elements have recently been found in the promoters three Gl cyclin genes. In addition, we have shown that SWI6 is required for the periodic expression of SWI4 and a large group of DNA synthesis genes that contain a different cell cycle regulated promoter element ACGCGTNA (or MCB). This leads us to view SWI6 as a critical link between all the known periodic transcription events that occur in late Gl, and suggests that the regulation of SWI6 activity may define the Gl/S transition. We propose to continue our efforts to understand the mechanisms of Start- dependent tran-scription, with a particular focus on the partners and activities of the SWI4 and SWI6 proteins. We will look for Start-dependent changes in phosphorylation of SWI4 and SWI6. We will also characterize suppressors of swi6 mutations that we have isolated, and identify other gene products that interact with SWI6. We have found that one important cause of Gl/S-specific transcription of HO and the CLN genes is the cell cycle regulation of SWI4 transcription. The essential upstream activation sequence (UAS) in the SWI4 promoter resides within a 140 bp DNA fragment. It is sufficient for cell cycle regulation and contains three MCB elements. Surprisingly, deletion of all three MCB elements does not eliminate cell cycle regulated transcription. We propose further studies of the SWI4 promoter to identify the novel cell cycle regulatory element. The 140 bp SWI4 UAS is also unusual in that it does not activate transcription in the reverse orientation. We propose to screen for mutants in the sequence responsible for this repression and then to determine if this sequence performs a boundary function in its normal chromosomal location, insulating the SWI4 promoter from upstream UAS's. If so, we will obtain mutations in the factors responsible for this boundary activity. Finally, we have generated a stable, SWI6-independent form of the SWI4 protein which causes high level CLN1 and CLN2 transcription throughout the cell cycle. This has a dominant negative effect on cell growth. We propose to characterize this growth defect and to isolate compensatory mutations and high copy suppressors of this phenotype. In so doing, we will identify other cellular factors involved in Gl cyclin metabolism and activation.